1. Field of the Invention
Embodiments of the invention relate to compounds, compositions and methods of using Heat Shock Proteins for delivery and internalization of signaling agents and therapeutics. For instance, Heat Shock Protein 70 bind to a LOX-1 receptor and signal the LOX-1 receptor to transduce the HSP and any attached moiety across the endothelium. Therefore embodiments of the invention are useful for delivering various agents one desires to have internalized into a cell such as therapeutics and contrast/imaging agents. The isolated peptides may be attached either directly or through a linker to an imaging agent or a therapeutic. The compositions are also useful in delivering biological molecules to sites in vivo having high concentrations of oxidized low-density lipoprotein receptor (LOX-1 or OLR-1), including macrophages and other inflammatory cells. The contrast/imaging agents may be selected for various imaging modalities, more particularly the moieties are useful for imaging sites vulnerable to plaques associated with inflammation, such as atherosclerosis. The compositions are useful for the diagnosis and monitoring of inflammation and diseases in which inflammation plays a role such as various cardiovascular diseases including but not limited to atherosclerosis, vulnerable plaque and coronary artery disease as well as rheumatoid arthritis.
2. Description of Related Art
HSPs (Heat Shock Proteins) are a family of highly conserved proteins found in the cells of all organisms, from bacteria to mammals. HSPs are required for cellular metabolism even in unstressed cells. They facilitate the synthesis, structure, transport, and other aspects of protein assembly such as helping newly synthesized polypeptides fold and thus prevent premature interactions with other proteins (i.e. act as chaperones). HSP expression increases in response to physiological stresses such as a rise in temperature, altered pH and oxygen deprivation. These stresses may result in a break down of three-dimensional structure or unfolding of a cell's proteins. If the stress is left unchecked the mis-folded or unfolding proteins form aggregates that may eventually kill the cell. HSPs bind to damaged proteins helping them refold into their proper shapes and/or prevent the damage from occurring.
EP 1 046 652 A1 discloses a fusion polypeptide composed of an extracellular domain of mammalian oxidized-LDL receptor (LOX-1) and a part of IgG, whereby the fusion polypeptide may be labeled with a labeling agent. Thus, the fusion polypeptide can be used to detect, quantify, separate, and purify oxidized LDL. The fusion polypeptides can not be used to detect or quantify LOX-1.
The TAT peptide sequence and the recently reported Antp internalization sequence have demonstrated internalization activity of various substrates both in vitro and in vivo. Neither has been demonstrated to target a specific receptor for the purpose of delivering a diagnostic contrast agent to a diseased area on the endothelium, though TAT peptide has been used to non-specifically deliver iron oxide nanoparticles into cells (Wunderbalinger, P. et al., Bioconjugate Chemisfry, 2002, 13, 264-8). A disadvantage of these schema is the non-specificity of the peptides for targeting of the contrast agent to the cells of interest. The non-specific delivery of contrast agent would significantly deride attempts to distinguish areas of diagnostic interest (i.e. atherosclerotic lesions) from other functional areas of the vasculature.
Cardiovascular diseases are the leading cause of death in the United States, accounting annually for more than one million deaths. Atherosclerosis is the major contributor to coronary heart disease and is a primary cause of non-accidental death in Western countries (Coopers, E. S. Circulation 1993, 24, 629-632; WHO-MONICA Project. Circulation 1994, 90, 583-612). It is well-documented that multiple risk factors contribute to atherosclerosis such as hypertension, elevated total serum cholesterol, high levels of low density lipoprotein (LDL) cholesterol, low levels of high density lipoprotein (HDL) cholesterol, diabetes mellitus, severe obesity, and cigarette smoking (Orford et al., Am. J. Cardiol. 2000, 86 (suppl.) 6H-11H). Considerable effort has been made in defining the etiology and potential treatment of atherosclerosis and its consequences, including myocardial infarction, angina, organ failure and stroke. To date, treatment of atherosclerosis focuses on lowering cholesterol levels and modifying lipids. However, recent studies indicate that 40% of deaths due to coronary disease occurred in men with total cholesterol levels of below 220 mg/dl. (Orford et al). There are many unanswered questions including how and when atherosclerotic lesions become vulnerable and life-threatening, the best point of intervention, and how to detect and monitor the progression of lesions.
Several invasive and noninvasive techniques are routinely used to image atherosclerosis and to assess the progression and stabilization of the disease. These include coronary angiography, intravascular ultrasound angioscopy, intravascular magnetic resonance imaging, and thermal imaging of plaque using infrared catheters. These techniques have been used successfully to identify vulnerable plaques. However, these techniques are generally invasive, requiring surgery, insertion of probes, cameras, or other invasive procedures. For instance, soluble markers, such as P-selectin, von Willebrand factor, Angiotensin-converting enzyme (C 146), C-reactive protein, D-dimer (Ikeda et al., Am. J. Cardiol., 1990, 65, 1693-1696), and activated circulating inflammatory cells are found in patients with unstable angina pectoris however, their presence cannot be used to locate the involved lesion. Temperature sensing elements contained in catheters have been used for localizing plaque on the theory that inflammatory processes and cell proliferation are exothermic processes and are described for example in U.S. Pat. No. 4,986,671 and U.S. Pat. No. 4,752,141. An angiogram reflects luminal diameter and provides a measure of stenosis with excellent resolution, however, it does not image the vessel wall or the various histopathological components.
Techniques currently available typically identify some of the morphological and/or functional parameters of atherosclerosis and provide qualitative or semiquantitative assessment of the relative risk associated with the disease. However, these diagnostic procedures are either invasive or yield little information on the underlying pathophysiology such as cellular composition of the plaque, and biological characteristics of each component in the plaque at the molecular level. For further discussion of inflammatory related conditions and diseases see co-pending U.S. application Ser. Nos. 10/691,532 and 10/691,533 which are hereby incorporated by reference in their entirety.
Oxidized LDL (oxLDL) is strongly implicated in the pathobiology of atherosclerosis. It is suspected that the lipid pool in atherosclerotic plaque is due to uptake of oxLDL, not native LDL. OxLDL is recognized by scavenger receptors on macrophages; uptake of large quantities of oxLDL by macrophages can give rise to foam cells which are an important component of atherosclerotic plaque. LOX-1 or lectin-like oxidized LDL receptor was recently identified as a receptor on endothelial cells for oxLDL; it mediates the internalization of oxLDL by endothelial cells and is distinct from macrophage scavenger receptors such as those described in WO 2002/06771, (Sawamura, T. Nature 1997 386:73-77). The amino acid sequence of LOX-1 is shown in FIG. 3. LOX-1 also is expressed on macrophages and may play a role in oxLDL recognition/internalization on these cells (Yoshida, H. et al., Biochem. J. 1998 334:9-13). LOX-1 is nearly undetectable in healthy human aorta samples but is found in atherosclerotic plaque, particular early lesions that are unlikely to be detectable by other means (Kataoka, H. et al., Circulation 1999 99:3110-3117). Recent work suggests that recognition of oxLDL by LOX-1 is a critical early step in expression of adhesion receptors on endothelial cells. These receptors are believed to be responsible for attracting monocytes to the early atherosclerotic plaque.
As such, a non-invasive method to diagnose and monitor various cardiovascular diseases (e.g., atherosclerosis, vulnerable plaque, coronary artery disease, renal disease, thrombosis, transient ischemia due to clotting, stroke, myocardial infarction, organ transplant, organ failure and hypercholesterolemia) is needed. The non-invasive method should yield information regarding the underlying pathophysiology of the plaque, such as the cellular composition of the plaque and biological characteristics of each component in the plaque at the molecular level.
The description herein of disadvantages and deleterious properties and/or results achieved with known products, methods, and apparatus, is in no way intended to limit the scope of embodiments of the invention. Indeed, the present embodiments of the invention may utilize one or more known products, methods, and apparatus without suffering from the described disadvantages and deleterious properties and/or results.